JPS61181393A - Production of xanthane type polysaccharides - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing polysaccharides by fermentation of carbohydrates using microorganisms. More particularly, the present invention relates to fermentation methods in emulsions.

Complex polysaccharides or biopolymers obtained by fermentation of carbohydrates under the action of bacteria of the genus Xanthomonas, Ars tetrabacter, Sufflerotium and other fungi are widely used industrially due to their thickening and thickening properties. There is.

The production of xanthan gum by aerobic fermentation in aqueous media is covered by numerous patents, such as U.S. Patent No. 000,790;
Same No. 3.020.206, Same No. 4591,060,
Same 5th. a 53.7 o s no. 4.119.5
No. 46, No. 4.154.654, No. 4.296,
No. 205, No. 4.377.637 and French Patent No. 2.414,555.

° At the end of production, the fermented juice contains about 15 to 50 polysaccharides.
It may contain 11/l. Industrially, it is very difficult to increase the concentration higher than 30 to 35 y/l without taking special measures. In fact, as polysaccharides are formed, the viscosity of the reaction medium increases so that oxygen transport is reduced and fermentation is inhibited. Even if the reactor is equipped with expensive and high-capacity stirring equipment, it is very difficult to ensure aeration of the reaction mass and sufficient brewing to allow an increase in the concentration of the polymer.

A recently proposed method for increasing gum concentration involves carrying out the fermentation with an aqueous nutrient medium emulsified or dispersed in oil to reduce the viscosity of the fermentation broth and facilitate oxygen transport. (European Patent Application No. 005
8364 and 0074775) 0 However, the first step of microbial growth is carried out in a substantially aqueous nutrient medium before oil is introduced to make the emulsion a water-in-oil emulsion. is preferable. Once a water-in-oil emulsion is formed, it becomes difficult to control the pH of the dispersed aqueous phase and the formation of by-product acid products can inhibit the growth of microorganisms. It is certainly possible to operate in a buffered medium, but the presence of binding substances can be detrimental to the quality of the solution obtained. Furthermore, stabilized water-in-oil emulsions are very difficult to dilute, which is a disadvantage if these emulsions are intended to be used directly, for example in oil field development.

The object of the present invention is to provide a fermentation method in which the growth of microorganisms in an emulsifying medium can be easily carried out.

Furthermore, it is an object of the present invention to realize stable emulsions of biopolymers that can contain up to about 60% biopolymers.

A feature of the process for the production of polysaccharides by fermentation of an aqueous nutrient medium using microorganisms according to the invention is that the oil is dispersed in an aqueous medium so that an oil-in-water emulsion is formed in which the fermentation takes place. be.

For the continuous aqueous phase, any fermentation medium described in known literature may be selected. For details, reference may be made, for example, to US Pat. No. 5,391,060, US Pat. Conventional media contain a source of carbohydrates (which may be sugars or other carbohydrates), a source of organic nitrogen and/or inorganic salts, and trace elements and, if desired, growth factors. The concentration of sugars such as glutenose or sucrose is 10-10011
/l and can even reach 150-20011/l.

The causative microorganism of e.g.
S MANUAL OF DETERMINATIV
E BACTERIOLOGY) (8th edition (1974) William N. Wilkins, Inc., Baltimore)
Carbohydrate-fermenting bacteria, such as Xanthomonas begonia x (Xanthomona@b
egonias)s Xanthomonas campestris (
Xanthomonas campestria) Xanthomonas carotae (Xanthomonas e
arotae)s Xanthomonas hedera (Xanth
omonas hadera), Xanthomonas incanae (Xanthomonas 1ncanae), Xanthomonas malbacarearum (Xanthomonas
amalvaeearum), Xanthomonas papaveri, Kola (Xanthomonas papaveri)
colm), Xanthomonas 7 azeoli (Xant
homonas phassoll), Xanthomonas bishi (Xanthomonas pint),
vasculorum), Xanthomonas vericadoria (Xanthomonas ma rieatorlm)
, Xanthomonas bitianis
j vitiana), Xanthomonas pelargonii (Xanthomonas pelargoniI)%
, Arthrobacter-like bacteria, especially Arthrobacter stabilis (Arthr).
obaetsr 5tab mouth 1m), Azotobacter
・Indicus (Azotobact・r amount ndleu
s), Agrobacterium
m) bacteria of the genus, in particular Agrobacterium radiobacter a
ctor), Agrobacterium rhizogenes (Agr
obacterium rhizogenea), Agrobacterium tumefaciens (Agrobac
terium tumefaeiens). Soufflerotium (Scl@rotlum) 31
Fungi belonging to the genus can also be used.

Among these microorganisms, it is preferable to use Xanthomonas campestris.

Regarding the oil phase, all mineral or vegetable oils that are immiscible with water, such as isoparaffins, odorless kerosene, high kerosene (
Hydrocarbons, seed oils, soybean oil, corn oil, sunflower oil, peanut oil, etc. (preferably 150-200"C or higher) can be used. The proportion of the oil phase is less than 30% of the total medium, preferably from 1 to A suitable amount is 18%, preferably 3 to 16%.

Dispersion and stabilization of the oil phase in the aqueous phase is easily achieved by the presence of a preferably nonionic surfactant whose HLB value is preferably 11 or less, more preferably 6 to 11. In this case, the above HLB values are obtained by using a single surfactant or a mixture of surfactants. The type and amount of surfactant necessary to obtain a continuous aqueous phase can be easily determined by one skilled in the art from the type of oil selected and its concentration in the medium.

Examples of nonionic surfactants include compounds obtained by condensation of alkylene oxide with an aliphatic organic compound or an alkyl aromatic organic compound. Suitable surfactants that do not inhibit the growth of microorganisms include alkylphenol/polyoxyethylene, polyoxyethylene/
It is an ester of alcohol, fatty acid/polyoxyethylene, polyoxyethylene/triglyceride, sorbitan/polyoxyethylene and fatty acid.

All surfactants can be used alone or in combination of two or more. Depending on the surfactant affinity, all or part of one or more surfactants can be introduced into the oil phase or the aqueous phase.

To carry out the process of the invention it is recommended to sterilize the oil and aqueous phases separately. After sterilization and cooling, the oil phase may be mixed with the aqueous phase in the fermenter, and each phase may be sterilized separately. It may also be introduced continuously into the fermenter under stirring. This immediately forms a stable emulsion. Inoculation and fermentation are then carried out in a customary manner with aeration and stirring. During the fermentation, the pH can be maintained at an optimum value by injection of alkaline or ammonia solutions. Similarly, the medium required for growth can be provided periodically or continuously.

Once the fermentation has ended, the emulsion can be used as is, according to the intended use, or the fermentation liquid can be left in its raw state by separating the aqueous phase from the oil phase by destabilization using methods known per se. Alternatively, the polysaccharide can be precipitated with the addition of a lower alcohol, such as isopropyl alcohol, with or without the addition of an inorganic salt. The precipitated polysaccharide is separated, washed away from the oil adsorbed on the fibers, then dried and optionally ground. A powder ready for use is thus obtained.

According to a particular embodiment of the method of the invention, the emulsion obtained after carrying out the fermentation step contains from 8 to 60% by weight of polysaccharides, preferably from 15 to 60% by weight, relative to the weight of the emulsion. It is concentrated by removing water until an emulsion is obtained. In this embodiment, the amount of oil used for fermentation has to be calculated according to the content of dry material at the end of fermentation.

It is preferred that the amount of oil in the final emulsion does not exceed 45% of the total medium.

Removal of water can be carried out, for example, optionally by pressure evaporation or distillation. A portion of the oil field may be left in an azeotrope with water or otherwise. Alternatively, ultra-filtration can be carried out using conventional methods and equipment, provided of course that a hydrophilic porous membrane is selected whose pores are extremely small and the passage of the biopolymer through the membrane is forced open. It can also be operated from here. Ultrafiltration can be used to maintain the emulsion in a continuous aqueous phase.

Concentration by ultrafiltration has the advantage of keeping the inorganic salt content of the aqueous phase close to that present in the initial fermentation broth, regardless of the polymer content. .

In another embodiment, the emulsion can also be concentrated first by ultrafiltration and then by other operations, such as evaporation or distillation.

Without departing from the scope of the invention, it is possible to add bactericides, enzymes, and other additives intended for specific uses to the resulting emulsion, as long as such additives do not impair the stability of the emulsion. I can do it.

The advantages of the process according to the invention over emulsion systems of the opposite type are, in particular:

It is possible to achieve microbial growth in the emulsifying medium at one normal reaction rate. Microbial growth is not disturbed by the emulsion.

Energy is saved during one growth phase and less power is consumed to stir the medium.

- The pH of the aqueous phase can be easily adjusted.

- Emulsions can be concentrated without phase inversion and without precipitating polysaccharides ◇ Oil-in-water emulsions obtained according to the method of the present invention can be used in construction, painting, cosmetics, plant hygiene, petroleum, etc. It is used in all applications requiring thickened aqueous fluids, such as in the industry. These emulsions have the advantage that they do not need to undergo phase inversion and can be used directly to prepare dilute aqueous solutions. They disperse very well in water and their rate of dissolution is at least equal to that of the initial fermentation broth. The emulsion is pumpable and highly stable during concentration. Since its viscosity is substantially lower than that of an aqueous solution containing an equal amount of biopolymer,
A flowable or pumpable emulsion with an increased concentration of polysaccharide in the aqueous phase can be obtained.

All of these advantages make the emulsions of the invention particularly suitable for use in the oil field or for preparing aqueous media intended for auxiliary oil recovery.

The following examples are intended to explain the invention in more detail, but the invention is not limited to these examples.

In each example, operations were generally performed as follows.

The mononutrient aqueous solution is charged into a fermenter and sterilized.

- Sterilize the oil, optionally containing surfactants, and, under stirring,
Inject at ambient temperature into the aqueous solution contained in the fermenter.

- Add nutrient broth MY Sacca 0-XjO11/ to this culture medium.
Inoculate Xanthomonas campestris pre-cultured with l.

Example 1 2101/l of preculture in a 201 volume fermenter.

An oil-in-water emulsion 147 containing 625% by volume of an oil phase and 9575% by volume of an aqueous phase is inoculated.

Composition of aqueous phase Dextrose 619/1 yeast extract
711/IN a* HP No. 0 2.55fi/
1(NH4)*POa '1.21/
lMg804・7H Mayuzumi 0 α5511/1 Composition of oil phase Fermentation conditions Temperature 28°C pH Adjusted to 7 by adding soda Stirring 400 rpm Aeration 0-17 hours α24VVM 17-23 hours α48V%'M 23-90 hours (L98VVM fermentation The progress was made to consume all the sugar.

Table 1 shows the changes in the specific resistance of the population and culture medium over the course of 90 hours.

The value of the active substance obtained is 4251 (3α39/kI), ie, a yield of 56% based on sugar.

The viscosity of the emulsion at the end of fermentation is aIPa, a (Pluck tweed warp 0°C - needle A4 - 50t/min) ◇ Example 2 Hydrocarbon gxsol D 100 (29% by volume) and the following composition Example 1 was repeated using an initial medium consisting of an aqueous phase (71% by volume).

Dextrose 649/yeast extract 9.24.9/tNaHPO41
08JF/l (NH4) t p O, 50811/1M, SO4
・7H,O (L3!5g/l No surfactant added.

The same operating conditions as in Example 1 were used except that ventilation was always performed with α98VVM. Fermentation took place for 85 hours,
At that stage sugar was no longer present.

The changes in resistivity of the population and medium are shown in Table 1.

Active substance 350I was obtained. That is, the yield based on sugar is StS%. The viscosity of the final emulsion is 112 Pa, s
(same conditions as Example 1).

Example 3 Aqueous phase 14. An initial oil-in-water emulsion is formed from J 251 and the oil phase α875.

Saccharose 851! /1 yeast extract 25.57771Mg5O, 7H1
Oα2811/1 The oil phase contains 70% by weight of seed oil and 30% by weight of ethoxylated nonylphenol (CEMULSOL NF2).

HLB value 9 The medium is inoculated as in Example 1.

Fermentation conditions Temperature 28℃ Adjusted pH to 7 by adding soda Stirring 0 to 17 hours 590 rpm 17 to 24 hours 500 rpm 24 to 84 hours 600 rpm Aeration 0 to 17 hours (L92VVM 17 to 84 hours t4VVM Average negative power < L 09 KW/min After 84 hours, 51 xanthan rubber per gram of media IJ
．． 9 was generated. Yield based on consumed sucrose 6
4% The consistency K of the medium (according to Ostwall F's law) is 37 P
a, m and pseudoplasticity index n are α195.

In comparison, polymers 4 & 5. ? The consistency of the aqueous fermentation broth containing /39 is 70 pm, s and the index n is 0.
It is 24.

Fermentation was continued for up to 113 hours, yielding 66 Ii/Iq of polymer.

The yield based on consumed sucrose is 85%.

Two fermentations were carried out in an oil-in-water emulsion containing 0.8751/ml of oil phase and 14125/ml of aqueous phase.

The composition of the medium is as follows.

Sucrose 8% Water-soluble corn extract (C8L) 4% MgSO4.7
H,0 (LO5% oil 5 5
% Blush active agent 15% In Example 4, the oil phase was a mixture of seed oil and ethoxylated natural fatty acids (
CEMULSOL A35-8FO8), and in Example 5, aliphatic carbonized tree leaves (EXSOL DAD) and ethoxylated nonylphenol (CEMULSOL NF2-CEMUL)
50150 mixture of SOL NF17).

The fermentation conditions are as follows.

Temperature 28℃ Ventilation Stirring 0-17 hours a 92 V'VM 590 rpm
After 17 hours 159 VVM 500 rpm After 41 hours 139 VVM 500 rpm (Example 4 600 rpm (Example 5)
47.411/ky 519/Jam Yield on saccharose 60% 64% using a preculture of Xanthomonas campestris 1- grown on nutrient broth MY sucrose 109/l
100 d of oil-in-water emulsion medium in a 0-Erlenmeyer flask was inoculated.

The aqueous phase common to each example is the same as the aqueous phase of Example 3.

The composition of the oil phase was varied. Each flask contains 95% 1%
of water phase and 5% by weight of oil phase. 5 flasks
The mixture was stirred using a rotary stirrer rotating at 22 Orpm with a width of c+n, and cultured at 280''C for 89 hours.

The results obtained are shown in Table 2.

Claims (8)

[Claims] (1) A process for the production of polysaccharides by fermentation of an aqueous nutrient medium using microorganisms, characterized in that the oil is dispersed in an aqueous medium such that an oil-in-water emulsion is formed in which the fermentation takes place. . (2) Process according to claim 1, characterized in that the proportion of the oil phase is less than 30% by weight, preferably from 1 to 18% by weight of the weight of the emulsion. (3) Process according to claim 2, characterized in that the proportion of the oil phase is 3 to 16% by weight of the weight of the emulsion. (4) According to any one of claims (1) to (3), characterized in that the oil-in-water emulsion is stabilized using at least one nonionic surfactant. Method described. (5) The method according to claim (4), characterized in that the HLB value of the surfactant or the mixture of surfactants is 11 or less, preferably 6 to 11. (6) The method according to any one of claims (1) to (5), wherein the microorganism is of the genus Xanthomonas. (7) Fermentation is carried out in an oil-in-water emulsion containing less than 30% by weight of oil phase based on the weight of the emulsion, and then after the production of polysaccharides, the emulsion is mixed with 8 to 60% by weight of polysaccharides. A process for fermentation of polysaccharides by fermentation of an aqueous nutrient medium using microorganisms, characterized by concentration by removal of water until an emulsion containing % is obtained. (8) Oil-in-water emulsion containing 3 to 60 parts by weight of polysaccharide.